Characterization of GRK2-catalyzed phosphorylation of the human substance P receptor in Sf9 membranes.
G protein-coupled receptor kinases (GRKs) phosphorylate agonist-occupied G protein-coupled receptors (GPCRs), resulting in GPCR desensitization. GRK2 is one of the better studied of the six known GRKs and phosphorylates several GPCRs. In a previous study, we documented that GRK2 and GRK3 phosphorylate purified and reconstituted rat substance P receptor (rSPR) [Kwatra et al. (1993) J. Biol. Chem. 268, 9161-9164]. Here, we characterize in detail GRK2-catalyzed phosphorylation of human SPR (hSPR) in intact membranes. GRK2 phosphorylates hSPR in urea-washed Sf9 membranes in an agonist-dependent manner with a stoichiometry of 19 +/- 1 mol of phosphate/mol of receptor, which increases slightly (1.3-fold increase) in the presence of G beta gamma. Kinetic analyses indicate that receptor phosphorylation occurs with a Km of 6.3 +/- 0.4 nM and a Vmax of 1.8 +/- 0.1 nmol/min/mg; these kinetic parameters are only slightly affected by G beta gamma [Km = 3.6 +/- 1.0 nM and Vmax = 2.2 +/- 0.2 nmol/min/mg]. The lack of a strong stimulatory effect of G beta gamma on GRK2-catalyzed phosphorylation of hSPR is surprising since G beta gamma potently stimulates GRK2-catalyzed phosphorylation of beta 2-adrenergic receptor and rhodopsin. Involvement of G beta gamma endogenously present in membranes is ruled out as a source of high levels of hSPR phosphorylation, since receptor phosphorylation was not affected by guanine nucleotides that suppress or enhance the release of endogenous G beta gamma. The present study determines, for the first time, the kinetics of phosphorylation of a receptor substrate of GRK2 in intact membranes. Further, our results identify hSPR as a unique substrate of GRK2 whose phosphorylation is strong even in the absence of G beta gamma.
Nishimura
K
K
Department of Anesthesiology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Warabi
K
K
Roush
E D
ED
Frederick
J
J
Schwinn
D A
DA
Kwatra
M M
MM
D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis / Q000187:drug effects; D002462:Cell Membrane; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism / Q000502:physiology; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D018040:Receptors, Neurokinin-1 / Q000819:agonists / Q000187:drug effects / Q000378:metabolism*; D018411:Spodoptera; D014508:Urea; D051552:beta-Adrenergic Receptor Kinases
D018040:Receptors, Neurokinin-1; D014508:Urea; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; D019204:GTP-Binding Proteins